Highly Efficient Targeted Mutagenesis in Mice Using TALENs

Abstract

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. GENETIC engineering of cells and organisms to create targeted mutants is a key technology for genetics and biotechnology. The ascent of the mouse as a mammalian genetic model is based on gene targeting through homologous recombination (HR) in embryonic stem (ES) cells (Capecchi 2005). Classical gene targeting via ES cells is a time-and labor-intense procedure that proceeds in the steps of vector construction, ES cell mutagenesis, chimera generation, and the transmission of mutant alleles through the germline (Hasty et al. 2000). Since the frequency of spontaneous HR in ES cells is low, it was a key finding that double-strand breaks (DSBs), created by sequence-specific nucleases, enhance local DNA repair by several orders of magnitude (Rouet et al. 1994). DSBs may be repaired through HR, using the sister chromosome as template or using gene targeting vectors that provide sequence homology regions flanking a desired genetic modification (Court et al. 2002;San Filippo et al. 2008). Alternatively, DSBs can be sealed by the nonhomologous end-joining (NHEJ) pathway that religates open ends without a repair template (Lieber 2010). By this means the DNA ends are frequently edited through the loss of multiple nucleotides, causing frameshift (knockout) mutations within coding regions. Targeted DSBs were first induced by zinc-finger nuclease (ZFN) fusion proteins that combine a DNA-binding domain made of zinc-finger motifs with the nuclease domain of FokI (Porteus and Carroll 2005). The application of ZFNs in one-cell embryos provided proof-of-principle for the direct mutagenesis of the mouse, rat...

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

“…We observed TALEN-mediated induction of NHEJ in 25.4% of the offspring independent of targeting construct integration. These results are in line with efficiencies reported for zinc-finger nucleases in mice [25][26][27] and rats 28 , as well as for TALEN-injections in murine oocytes 6,12,13,29 . Newly established methodology using the CRISPR/Cas9 system to induce DNA double-strand breaks and facilitate homologous recombination 21,22 certainly raises high expectations that this system could also be used to mediate homologous recombination of larger donor plasmids directly in oocytes 22 .…”

“…11). While this manuscript was in preparation such findings have been extended to the mouse using a similar strategy for homologous recombination in mice that utilizes either short ssDNA donor templates or donor vectors to correct a single base mutation 12,13 . This work demonstrated that a donor vector with long homology regions, similar to the one used for this study, can be used for homology-directed repair in oocytes to exchange single base mutations.…”

“…Gene targeting via non-homologous end-joining (NHEJ) has also been suggested to generate combined reporter and knockout alleles 9 . Combination of TALEN-based gene targeting with insertion of a few base pairs or transgenes using small ssDNA oligonucleotides or donor vectors with longer homologous arms was initially reported for zebrafish 10,11 and later extended to other animal models including mouse 12,13 . The mouse remains the most widely used animal model in the study of human biology and diseases.…”

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases

“…Rab3 (Wada et al, 1997) 1 (Figueiredo et al, 2008) 2 Rab27 (Mahoney et al, 2006) 4 (Figueiredo et al, 2008) 2 (Yoshimura et al, 2010) 1 Rab3, Kif1a, Kif1b, DR4, DR5, TNFR1, JNK3, LRDD, IA-2 (Marat et al, 2011) Rabconnectin-3 (Kawabe et al, 2003) Madd-null mice (Tanaka et al, 2001) C. elegans aex-3 mutant (Iwasaki et al, 1997) DENN-related FLCN (201163) folliculin Rab35 (Nookala et al, 2012) 1 GAP for RagC/D GTPase (Tsun et al, 2013) Birt-Hogg-Dubé syndrome Flcn-, Fnip1-, Fnip2-null mice (Schmidt and Linehan, 2015) FNIP1 ( C9ORF72 (427370) DENNL72 ND Rab1, Rab5, Rab7, Rab11, ubiquilin-1/2, hnRNPA1, hnRNPA2/B1, actin (Farg, et al, 2014) C9orf72-null mice (Panda et al, 2013;Koppers et al, 2015) Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (Lattante et al, 2015) SMCR8 ( …”

Transgenesis in farm animals-A review

“…Like ZFN, Tale repeats are linked together in order to recognize specific DNA sequences. TALEN has been rapidly and widely used to perform precise genome editing in a wide range of cell types and organisms, including rats, mice (Panda et al, 2013), human and livestock (Moghaddassi et al, 2014).…”